Electrophoresis approaches have been long used in the separation and analysis of samples for variety of purposes, such as for identifying a particular substance or for determining the size and type of molecules in a solution. For example, a variety of molecular biology applications have employed electrophoresis to separate proteins or nucleic acids, determine molecular weight, and/or prepare samples for further analysis. In these and other applications, electrophoresis generally involves the movement of an electrically-charged substance (e.g., molecules or ions) under the influence of an electric field. This movement facilitates the separation of a sample from other samples or substances. Once separated, the sample is readily analyzed using an optical or other approach.
A variety of electrophoresis-based approaches are used in connection with different applications to suit the particular needs of the analysis that to be performed. One type of electrophoresis is capillary electrophoresis (CE), which separates ionic samples using charge and frictional forces, generally within a small channel or capillary, using the size-to-charge ratio of the samples to facilitate their respective separation. One particular type of CE that has been the subject of extensive research in recent years is on-chip CE, wherein a small channel or capillary is formed on a microchip. On-chip CE has been useful to facilitate rapid sample separation and reduced sample volumes, and has further been readily integrated with other microfluidic functions. However, the limit of detection (LOD) of on-chip CE systems can be limited by associated small sample volumes and the shallow depth of etched channels (e.g., 10-20 μm), which limits the path length available for imaging a sample or samples in the channel.
In some applications, sample stacking approaches are used with on-chip CE to improve the sensitivity of sample analysis, facilitating the use of less sensitive detection approaches such as electrochemical detection or ultraviolet (UV) absorption. Stacking also facilitates robust injection schemes because the preconcentration of samples, as is associated with stacking, reduces the effective sample injection width (e.g., the width of sample material in a capillary or microchannel).
One type of on-chip stacking involves electromigration-based processes, such as field-amplified sample stacking (FASS), large volume sample stacking, and isotachophoresis (ITP). Electromigration-based sample stacking leverages spatial gradients of electrophoretic velocity of sample analytes as effected by gradients in ion density, mobility, and/or solvent viscosity. In FASS, signal enhancement factors have been generally limited to 1,000-fold using free-standing capillaries, and 100-fold using microchips, prior to demonstration of 1000-fold on-chip FASS. In ITP, charged particles are separated using an electric field to create boundaries or interfaces between materials (e.g., between the charged particles and other materials in a solution). ITP has been combined with CE as a robust, pre-separation sample stacking approach, has been demonstrated using rare earth metals and simple acids, and has been integrated with on-chip CE approaches. ITP generally uses multiple electrolytes, where the electrophoretic mobilities of sample ions are less than that of a leading electrolyte (LE) and greater than that of a trailing electrolyte (TE) that are placed in a microchannel or capillary. Individual species of a sample form narrow zones between the LE and TE and migrate with the same velocity (i.e., “isotacho” means equal velocity). With judicious choice of LE and TE chemistry, ITP is fairly generally applicable, can be accomplished with samples initially dissolved in either or both the TE and LE electrolytes, and (unlike FASS) does not require very low electrical conductivity background electrolytes.
While ITP has been readily implemented and useful for many applications, its implementation has been challenging for a variety of reasons. For instance, one challenging aspect of ITP has been that it requires significant knowledge of the electrophoretic mobilities of sample ions, prior to analysis, to ensure that the TE and LE electrolytes are appropriately chosen. That is, the nature of the TE and LE are relatively important in these prior approaches, to achieve adequate sample separation. In addition, controlling the placement and progression of materials in fluid channels in a manner that facilitates desirable analysis has also been challenging. Moreover, integrated ITP and CE approaches have generally required several steps in the separation and analysis process, and as such can be cumbersome and time consuming. These and other characteristics have been challenging to electrophoresis, isotachophoresis and related applications.